1. Field of the Invention
This invention relates to a method for producing tissue type plasminogen activator (hereinafter referred to as tPA) produced by human normal cells.
tPA which is produced and excreted by vascular endothelial cells and various tissue cells lyses fibrin clots, namely thrombi. Thus, tPA is effective as a thrombolytic agent.
2. Description of the Prior Art
tPA has two molecular forms, single-chain tPA and double-chain tPA. Thrombolytic activity of double-chain tPA is higher than that of single-chain tPA. So-called tPA has been conventionally developed as the sole double-chain form or the mixture form of the double-chain and the single-chain form.
Double-chain tPA has high fibrinolytic activity, and it is highly possible that double-chain tPA activates plasminogen not in thrombi where fibrinolytic effect is expected but in the blood stream, which often causes clinical bleeding (Japenese Patent Laid-open Pub. No. 118717/1984).
On the other hand, single-chain tPA which is considered to be a precursor of double-chain tPA has high affinity to fibrin and is quickly converted to double-chain tPA once bound to fibrin.
Accordingly, single-chain tPA maximally exhibits plasminogen activity at clotting sites.
Thus, thrombolytic activity of single-chain tPA is relatively low and not exhibited in the blood stream. In consequence, for clinical use, single-chain tPA is in greater demand than double-chain tPA, and an effective method for the production of single-chain tPA is highly requested.
However, in production of single-chain tPA using cells, there is a problem that proteolytic enzymes contained in a medium or produced by the cells (mostly considered to be plasmin or trypsin) convert single-chain tPA to double-chain tPA during production processes, which interferes with effective production of single-chain tPA.
Relevant methods known to solve such problem are described below.
They are a method in which cultivation and subsequent steps are carried out in the presence of aprotinin (European Patent Publication No. 41766), a method in which trypsin inhibitor induced by soybeans or aprotinin is added in the culture medium for tPA-producing cells (Japanese Patent Laid-open Pub. No. 118717/1984), a method in which cultivation or induction production is carried out in a medium supplemented with aprotinin or benzamidine (Japanese Patent Laid-Open Pub. No. 19486/1986) and a method in which sole single-chain tPA is produced by adding aprotinin or 6-aminocapronic acid in a purification process (Biochem. Biophys. Acta 719(2), 318-328, 1982).
Furthermore, particularly in the case where adhesive cells are used, there is a difficult problem that the cells are detached from the wall of a container or the surface of beads during cultivation. To solve this problem, removal of plasmin from the serum and addition of aprotinin are suggested (Kaufman Molecular and Cellular Biology, 5, pp1750).
However, there are many difficulties in practicing these conventional methods on industrial scale. For example, aprotinin to be used is quite expensive. And removal of plasmin from the serum requires complicated processes.